Ali El-Tayeb

Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors

Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of β-adrenergic receptors. Because BAT therapies based on cold exposure or β-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells—so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine–A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.

Selective Activation of Adenosine A2A Receptors on Immune Cells by a CD73-Dependent Prodrug Suppresses Joint Inflammation in Experimental Rheumatoid Arthritis

Phosphorylated adenosine A2A receptor agonists suppressed inflammation in a model of arthritis without A2A-mediated vasodilatory side effects. Separating the Wheat from the Chaff Extolling the virtues of simple building design, the modern architect Ludwig Mies van der Rohe famously declared that “less is more,” a philosophy that applies to modern drug design as well. Because simpler drugs have fewer side effects, the promise of adenosine A2A receptor agonists as therapeutics would grow if one could only separate their anti-inflammatory and vasodilator functions. Now, Flögel et al. built an adenosine A2A receptor agonist (chet-AMP) that displays only the anti-inflammatory function in an animal model of rheumatoid arthritis. How the authors accomplished this feat lies “in the details,” to again paraphrase van der Rohe. To isolate the anti-inflammatory effects of A2AR, Flögel et al. synthesized a prodrug that required, for its activation, the presence of ecto-5′-nucleotidase (CD73), which is mainly found on endothelial and immune cells. Using 19F magnetic resonance imaging to track inflammation noninvasively over time, the authors showed that chet-AMP, but not chet-adenosine, reduced inflammation in a mouse model of collagen-induced arthritis. This effect was dependent on the presence of both CD73 and A2AR, and no vasodilation was observed until drug concentrations were increased 100-fold. Physicians most often use corticosteroids to treat inflammatory conditions, but these drugs, although effective, can cause serious complications. By simplifying delivery of a drug only to the sites where it is needed most, Flögel et al. quell inflammation and avoid an undesirable side effect. Adenosine A2A receptor (A2AR) agonists are both highly effective anti-inflammatory agents and potent vasodilators. To separate these two activities, we have synthesized phosphorylated A2AR agonists (prodrugs) that require the presence of ecto-5′-nucleotidase (CD73) to become activated. In the model of collagen-induced arthritis, 2-(cyclohexylethylthio)adenosine 5′-monophosphate (chet-AMP), but not 2-(cyclohexylethylthio)adenosine (chet-adenosine), potently reduced inflammation as assessed by fluorine-19 (19F) magnetic resonance imaging and by histology. The prodrug effect was blunted by inhibition of CD73 and A2AR. The selectivity of drug action is due to profound up-regulation of CD73 and adenosine A2AR expression in neutrophils and inflammatory monocytes as found in recovered cells from the synovial fluid of arthritic mice. Plasma chet-adenosine was in the subnanomolar range when chet-AMP was applied, whereas concentrations required for vasodilation were about 100 times higher. Thus, chet-AMP is a potent immunosuppressant with negligible vasodilatory activity. These data suggest that phosphorylated A2AR agonists may serve as a promising new group of drugs for targeted immunotherapy of inflammation.

Ecto-5′-Nucleotidase (CD73)-Mediated Formation of Adenosine Is Critical for the Striatal Adenosine A2A Receptor Functions

Adenosine is a neuromodulator acting through inhibitory A1 receptors (A1Rs) and facilitatory A2ARs, which have similar affinities for adenosine. It has been shown that the activity of intracellular adenosine kinase preferentially controls the activation of A1Rs, but the source of the adenosine activating A2ARs is unknown. We now show that ecto-5′-nucleotidase (CD73), the major enzyme able to convert extracellular AMP into adenosine, colocalizes with A2ARs in the basal ganglia. In addition to astrocytes, striatal CD73 is prominently localized to postsynaptic sites. Notably, CD73 coimmunoprecipitated with A2ARs and proximity ligation assays confirmed the close proximity of CD73 and A2ARs in the striatum. Accordingly, the cAMP formation in synaptosomes as well as the hypolocomotion induced by a novel A2AR prodrug that requires CD73 metabolization to activate A2ARs were observed in wild-type mice, but not in CD73 knock-out (KO) mice or A2AR KO mice. Moreover, CD73 KO mice displayed increased working memory performance and a blunted amphetamine-induced sensitization, mimicking the phenotype of global or forebrain-A2AR KO mice, as well as upon pharmacological A2AR blockade. These results show that CD73-mediated formation of extracellular adenosine is responsible for the activation of striatal A2AR function. This study points to CD73 as a new target that can fine-tune A2AR activity, and a novel therapeutic target to manipulate A2AR-mediated control of striatal function and neurodegeneration.

N-substituted phenoxazine and acridone derivatives: structure-activity relationships of potent P2X4 receptor antagonists.

P2X4 receptor antagonists have potential as drugs for the treatment of neuropathic pain and neurodegenerative diseases. In the present study the discovery of phenoxazine derivatives as potent P2X4 antagonists is described. N-Substituted phenoxazine and related acridone and benzoxazine derivatives were synthesized and optimized with regard to their potency to inhibit ATP-induced calcium influx in 1321N1 astrocytoma cells stably transfected with the human P2X4 receptor. In addition, species selectivity (rat, mouse, human) and receptor subtype selectivity (versus P2X1,2,3,7) were investigated. The most potent P2X4 antagonist of the present series was N-(benzyloxycarbonyl)phenoxazine (26, PSB-12054) with an IC(50) of 0.189 μM and good selectivity versus the other human P2X receptor subtypes. N-(p-Methylphenylsulfonyl)phenoxazine (21, PSB-12062) was identified as a selective P2X4 antagonist that was equally potent in all three species (IC(50): 0.928-1.76 μM). The compounds showed an allosteric mechanism of action. The present study represents the first structure-activity relationship analysis of P2X4 antagonists.

2-Amino-5-benzoyl-4-phenylthiazoles: Development of potent and selective adenosine A1 receptor antagonists

A series of 2-amino-5-benzoyl-4-phenylthiazole derivatives was investigated in radioligand binding studies at adenosine receptor (AdoR) subtypes with the goal to obtain potent and A(1)-selective antagonists. Acylation of the 2-amino group was found to be crucial for high A(1) affinity. The best compound of the present series was 2-benzoylamino-5-p-methylbenzoyl-4-phenylthiazole (16 m) showing a K(i) value of 4.83 nM at rat and 57.4 nM at human A(1) receptors combined with high selectivity versus the other AdoR subtypes. The compound behaved as an antagonist in GTP shift assays at A(1) receptors. Compound 16 m may serve as a new lead structure for the development of second-generation non-xanthine-derived A(1) antagonists which have potential as novel drugs.

Development of Polar Adenosine A2A Receptor Agonists for Inflammatory Bowel Disease: Synergism with A2B Antagonists

Adenosine A2A receptor agonists for the local treatment of inflammatory bowel disease (IBS) were designed and synthesized. Polar groups were introduced to prevent peroral absorption and subsequent systemic, e.g., hypotensive, side effects. 4-(2-{6-Amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-9H-purin-2-ylthio}ethyl)benzenesulfonic acid (7, PSB-0777) was selected for further evaluation in rat ileum/jejunum preparations in ex vivo experiments. Compound 7 significantly improved impaired acetylcholine-induced contractions induced by 2,4,6-trinitrobenzenesulfonic acid and showed synergism with an A2B-selective antagonist. Thus, nonabsorbable, locally active A2A agonists, as a monotherapy or in combination with an A2B antagonist, may be an efficient novel treatment for IBS, preventing the severe systemic side effects of known A2A agonists.

Carbamazepine derivatives with P2X4 receptor-blocking activity

Antagonists for the P2 receptor subtype P2X4, an ATP-activated cation channel receptor, have potential as novel drugs for the treatment of neuropathic pain and other inflammatory diseases. In the present study, a series of 47 carbamazepine derivatives including 32 novel compounds were designed, synthesized, and evaluated as P2X4 receptor antagonists. Their potency to inhibit ATP-induced calcium influx in 1321N1 astrocytoma cells stably transfected with the human P2X4 receptor was determined. Additionally, species selectivity (human, rat, mouse) and receptor subtype selectivity (P2X4 vs P2X1, 2, 3, 7) were investigated for selected derivatives. The most potent compound of the present series, which exhibited an allosteric mechanism of P2X4 inhibition, was N,N-diisopropyl-5H-dibenz[b,f]azepine-5-carboxamide (34, IC50 of 3.44μM). The present study extends the so far very limited knowledge on structure-activity relationships of P2X4 receptor antagonists.

Synthesis of BODIPY Derivatives Substituted with Various Bioconjugatable Linker Groups: A Construction Kit for Fluorescent Labeling of Receptor Ligands

The goal of the present study was to design small, functionalized green-emitting BODIPY dyes, which can readily be coupled to target molecules such as receptor ligands, or even be integrated into their pharmacophores. A simple two-step one-pot procedure starting from 2,4-dimethylpyrrole and ω-bromoalkylcarboxylic acid chlorides was used to obtain new ω-bromoalkyl-substituted BODIPY fluorophores (1a–1f) connected via alkyl spacers of different length to the 8-position of the fluorescent dye. The addition of radical inhibitors reduced the amount of side products. The ω-bromoalkyl-substituted BODIPYs were further converted to introduce various functional groups: iodo-substituted dyes were obtained by Finkelstein reaction in excellent yields; microwave-assisted reaction with methanolic ammonia led to fast and clean conversion to the amino-substituted dyes; a hydroxyl-substituted derivative was prepared by reaction with sodium ethylate, and thiol-substituted BODIPYs were obtained by reaction of 1a–1f with potassium thioacetate followed by alkaline cleavage of the thioesters. Water-soluble derivatives were prepared by introducing sulfonate groups into the 2- and 6-position of the BODIPY core. The synthesized BODIPY derivatives showed high fluorescent yields and appeared to be stable under basic, reducing and oxidative conditions. As a proof of concept, 2-thioadenosine was alkylated with bromoethyl-BODIPY 1b. The resulting fluorescent 2-substituted adenosine derivative 15 displayed selectivity for the A3 adenosine receptor (ARs) over the other AR subtypes, showed agonistic activity, and may thus become a useful tool for studying A3ARs, or a lead structure for further optimization. The new functionalized dyes may be widely used for fluorescent labeling allowing the investigation of biological targets and processes.

Role of extracellular cysteine residues in the adenosine A2A receptor

The G protein-coupled A2A adenosine receptor represents an important drug target. Crystal structures and modeling studies indicated that three disulfide bonds are formed between ECL1 and ECL2 (I, Cys712.69-Cys15945.43; II, Cys743.22-Cys14645.30, and III, Cys773.25-Cys16645.50). However, the A2BAR subtype appears to require only disulfide bond III for proper function. In this study, each of the three disulfide bonds in the A2AAR was disrupted by mutation of one of the cysteine residues to serine. The mutant receptors were stably expressed in Chinese hamster ovary cells and analyzed in cyclic adenosine monophosphate (cAMP) accumulation and radioligand binding studies using structurally diverse agonists: adenosine, NECA, CGS21680, and PSB-15826. Results were rationalized by molecular modeling. The observed effects were dependent on the investigated agonist. Loss of disulfide bond I led to a widening of the orthosteric binding pocket resulting in a strong reduction in the potency of adenosine, but not of NECA or 2-substituted nucleosides. Disruption of disulfide bond II led to a significant reduction in the agonists’ efficacy indicating its importance for receptor activation. Disulfide bond III disruption reduced potency and affinity of the small adenosine agonists and NECA, but not of the larger 2-substituted agonists. While all the three disulfide bonds were essential for high potency or efficacy of adenosine, structural modification of the nucleoside could rescue affinity or efficacy at the mutant receptors. At present, it cannot be excluded that formation of the extracellular disulfide bonds in the A2AAR is dynamic. This might add another level of G protein-coupled receptor (GPCR) modulation, in particular for the cysteine-rich A2A and A2BARs.

Characterization of P2X4 receptor agonists and antagonists by calcium influx and radioligand binding studies

Graphical abstract Figure. No Caption available. ABSTRACT Antagonists for ATP‐activated P2X4 ion channel receptors are currently in the focus as novel drug targets, in particular for the treatment of neuropathic and inflammatory pain. We stably expressed the human, rat and mouse P2X4 receptors in 1321N1 astrocytoma cells, which is devoid of functional nucleotide receptors, by retroviral transfection, and established monoclonal cell lines. Calcium flux assay conditions were optimized for high‐throughput screening resulting in a Z′‐factor of >0.8. The application of ready‐to‐use frozen cells did not negatively affect the results of the calcium assays, which is of great advantage for the screening of compound libraries. Species differences were observed, the rat P2X4 receptor being particularly insensitive to many ATP derivatives. Membrane preparations of the cell lines showed high levels of specific [35S]ATP&ggr;S binding with low nonspecific binding (<5% of total binding), while non‐transfected cells were devoid of specific binding sites for the radioligand. Conditions were employed which allow binding studies to be performed at room temperature. While a variety of nucleotide‐derived agonists and the antagonist TNP‐ATP displaced [35S]ATP&ggr;S from its binding site at human P2X4 receptors, the non‐nucleotidic antagonists paroxetine and 5‐BDBD did not compete with radioligand binding and were therefore characterized as allosteric antagonists. Homology modeling was applied to find an explanation for the observed species differences.